The invention relates in general to a method and apparatus for processing images of a tissue sample microarray made up of a plurality of tissue microarray dots using an optical microscope. More particularly, the invention relates to a method and apparatus for capturing tissue sample images from the tissue sample microarray, indexing such images and manipulating and transmitting them.
It is known that in the treatment and prevention of cancers it is often important periodically to examine persons at risk for cancer. In some instances it may be necessary to biopsy tissue from such persons. As medical care has become available to more people and as the need for such increased vigilance has been recognized, the number of biopsies has increased.
One of the problems with evaluating biopsy materials is that in most cases microscopic evaluation of cellular structure and tissue architecture has been important in making determinations as to whether cellular changes have occurred in tissues indicative of cancer or whether cancer is actually present. In the past, such determinations have been made by employing microscopic examinations of tissues and associated cellular structures.
A number of techniques have been developed, including techniques developed by the present inventors, for providing greater accuracy and throughput for such biopsy systems.
In one such system tissues from a particular patient which have been stained are positioned from a particular patient on a microscope slide and are imaged by a light microscope. The images are captured and digitized in a tiled format. The tiles can be reassembled substantially in real time to create pan and scan images of large amounts of tissue at high magnification while simultaneously providing a second digitized image of tissue at low magnification to provide a guide to regions of interest of the pathologist. This system has proven to be a boon to pathologists.
Improvements made upon that system, such as that disclosed in U.S. Pat. No. 6,031,930 to Bacus et al., are directed to further enhancements of the microscopic image examination in that detailed features of the morphometry of images of microscopic objects, such as cells, cell fragments, and the like, are made more easy. Statistical measures are applied which are highly discriminating for neoplasia across diverse tissue types, such as breast, colon, cancer, cervical tissue, and the like. In addition, such systems are valuable in providing assaying functions for different carcinogens and chemopreventive agents.
More specifically, such systems use microscopic images of stained neoplastic tissue sections which are microscopically scanned to provide electronic or digitally recorded. Morphometric features of tissue sample images are measured in first unit values and texture measurements of the tissue samples, such as a Markovian texture measurement, are also made. The respective results are recorded on a grading common scale so that progression of cancer can be ascertained relative to normal tissue.
An additional advance has been made, as exemplified by U.S. Pat. No. 6,101,265 to Bacus et al. Bacus et al. disclose the use of an imaging system which can scan stained tissue samples on microscope slides and generate tiled images thereof. The system also provides low and high magnification image pan and scan capability both locally and remotely. Typical magnifications are 1.25 power, 4 power, 20 power, and 40 power. This allows a pathologist at a remote site to be able to examine a complete and accurate magnified record of the tissue. This can occur over a packet network, such as the Internet or the like, without the need for a wide-band, high-speed transmission, such as a television line.
Despite each of the advantages which have been provided by the previous systems, they still have some drawbacks when presented with newer technologies for rapid assay of large amounts of tissue. Recently, molecular profiling of tissue specimens has come into wider use. This process has to do with the discovery of new genes and targeting genetic probes for attachment to particular tissue regions and molecules such as epitopes. Pharmaceutical companies and researchers in the biological sciences are interested in developing antibody-based probes using standard antibody staining reactions in order to detect molecular abnormalities on the surfaces of cells.
In order to assay such wide collections of patients, it is necessary to collect large amounts of data from the patients. It is known to prepare tissue sample microarrays which consist of a plurality of circular sections of tissue drawn from a variety of persons or sampling sites and placed on a single microscope slide. Such samples are prepared by taking a very small diameter punch, removing punch cores of tissue and placing them into open columns in a paraffin block which open columns are arranged in a grid type array which may for instance have two to three hundred columns available. The total size of the block is small enough that an end section of the block would conveniently fit on a microscope slide under a cover glass or cover slip. Once the columns of tissue are placed within the block, the block is further treated so that the paraffin invades the tissue to provide a typical paraffin biological specimen. The block may be sectioned using separate microtome sectioning techniques and the sections with the two to three hundred circular tissue sample xe2x80x9cdotsxe2x80x9d may be placed on a microscope slide.
The slide may be subjected to staining and other antibody treatment and has the particular advantage that all two to three hundred of the specimens in the microarray are subjected to simultaneous and identical staining conditions, temperature conditions, and the like, so that variables need not be controlled for between patients who are being examined and a standardized treatment as applied to the tissue.
One of the problems, however, with such microarray-based assays is that the slides must be processed by hand. A microscopic determination must be made of characteristics of each of the tissue samples. At times tissue dots may fall off the slides opening up voids in the array or grid. It is easy for researchers who are examining the slides to lose track of which piece of tissue is being examined.
Although the microarray staining techniques have provided a considerable advantage in speeding up molecular assaying, the analysis of such results continues to be time-consuming and may be subject to more increased error than other types of assay systems.
What is needed then is a system which would provide for rapid assay of a microarray by an operator so that the advantages of bulk microarray treatment techniques can be fully realized.
A method and apparatus for processing an image of a tissue sample microarray include placing a plurality of tissue samples in an array or a rectangular grid on a conventional glass microscope slide. The tissue samples, which have been sectioned and form the array, may then be treated simultaneously as by staining the entire slide with a suitable biological stain for providing contrast to image various structures within the tissues or cells making up the multiple samples of the microarray. The advantage of doing this is that it allows multiple samples taking from multiple patients to be treated in substantially the same way and reduces the number of controls which must be put in place during the experiment.
The tissue sample microarray is prepared by taking a core or xe2x80x9cpunchxe2x80x9d of tissue from a tissue sample the core may have a diameter of a millimeter or less and the cored tissue is inserted into an open channel which is a right circular cylinder channel which is one of a rectangular array of channels formed in a paraffin block. The paraffin block is then treated so that the paraffin enters the tissue which is placed into each of the channels. The block may then be sectioned by a microtome as are other parafin block tissue samples. The microtome section may then be lifted and then placed on a microscope slide in a conventional manner. In this manner one or more microarrays may be placed on a single microscope slide. The microarrays typically have two to three hundred dots of tissue thereon, there may be up to six microarrays on a slide for over one thousand tissue samples on the slide which are subjected to substantially the same staining treatment. After staining, washing and the like are completed a cover slip is placed on the slide to make the slide permanent.
The slide may be placed on a movable stage of a scanning microscope. The microscope allows the user to take a low magnification image of a plurality of tissue dots in the microarray grid. The user may then identify corners of a scanned pattern. The scan pattern is usually rectangular and encompasses a subset of the tissue dots which are to be scanned.
An imager, optically connected to receive images from the microscope, generates a digital image which is fed to a processor and displayed on a display. The displayed image identifies the four corners of the grid to be scanned. It then shows target points at what would be expected to be the center points of the tissue dots in the grid array. Sometimes the dots are not precisely registered with uniform center-to-center distances within the array. Some tissue dots even may be missing. The user is then prompted with a cross-hair in each of the dots. Through the use of a mouse-select and drag operation the user can re-center the dot center point for the scan within each dot.
Once this has been done tiling grids are constructed and displayed over each of the images of the selected tissue dots. The tiling grids may typically include three rows and three columns of rectangular image tiles per tissue dot image. Tiling does not take place in the substantially empty or dot-free portions of the microarray.
The user may then command the computer to tile at least a portion of the dot images in the microarray. This consists of the microscope stage receiving commands at its X and Y stepper motors and then moving its stage and the slide it carries to a first position in a particular tile grid array of a selected dot image. A first dot image tile is captured. The stage is moved to the next tile position for that tissue dot and the next tile image is captured. In this manner all of the high magnification tile images at 20 to 40 power for that particular dot are captured. After completion of the capture of the first set of tile images for that dot additional capture takes place at successive tissue dots until the tiled images of all tissue dots within the selected tissue dot sub-array are captured.
After the dot images are captured data structures are set up associating block identification, user identification and patient identification with each of the tiled dot images. The specific identifying information at the block, user and patient levels may be inserted by the operator of the microscope at the time of dot image capture or later. In this way the tiled tissue dot microarray images are directly associated with the tissue dots in storage so that even if the original tissue microarray slide is lost the visual information and identifying information associated with the project would not be lost.
It is a principal aspect of the present invention to provide an image processing system which rapidly and completely scans and captures low magnification and high magnification images of a microarray of tissue specimens.
It is another aspect of the present invention to provide a method and apparatus for associating each of the images of the tissue samples or tissue dots within the microarray with a unique identifier so that the dot images can later be identified and accessed and be quickly and easily evaluated.
Other aspects and advantages of the present invention will become obvious to one of ordinary skill in the art upon appraisal of the remainder of the specification in light of the accompanying drawings and claims.